PIK3CA Hotspot Mutations P.H1047R And P.H1047L Sensitize Thymoquinone Treatment On Regulation Of PI3K/Akt1 Pathway In Breast Cancer

Objective: 1.Using PIK3CA/ΔNp63α complex as tumor activation inhibition domain,bioinformatics analysis and in vitro experiments were used to analyze the hotspot mutations of PIK3 CA in breast cancer and the intrinsic molecular mechanism of downstream pathway activation after mutations.2.To study the known inhibitory effect of thymoquinone（TQ）on PI3K/Akt1 pathway in breast cancer.3.To explore whether TQ can inhibit the activation of two hotspot mutations of PIK3 CA in PI3K/Akt1/MDM2 pathway in breast cancer.Methods: 1.NCBI program was used to analyze whether PIK3 CA gene and amino acid sequence were conserved in different substances;The Kaplan-Meier Plotter was used to analyze the recurrence free survival（RFS）in order to understand the correlation between the high expression of PIK3 CA and RFS in breast cancer.The mutation data of PIK3 CA in breast cancer patients were further analyzed from the “Cancer Browser” database to determine whether there were hotspot mutations.2.Cloning of expression vector of PIK3 CA gene and its mutant: we first obtained three plasmids,p HAGE-PIK3CA-WT,p HAGE-p.H1047 L and p HAGE-p.H1047 R from“Addgene” official website.After designing primers,we amplified our target fragments by PCR for further molecular cloning,and then loaded the three target fragments into the vector of pc DNA^TM5/FRT/TO/2ⅹFLAG.The PCR amplification,double enzyme digestion and sequencing were used to verify whether the clone is successful.3.The cloned recombinant plasmids were transfected into BT-549 and He La cell lines by Lipofectamine.Western blot（WB）was used to verify whether the recombinant plasmids could successfully express FLAG tagged protein,and the expression levels of p-Akt1 and total Akt1 were detected.4.Bioinformatics software was used to analyze two hotspot mutations of PIK3 CA in PIK3CA/ΔNp63α complex: p.H1047 R and p.H1047 L,and compared with PIK3 CA wild type（WT）to explore the activation mechanism of PI3K/Akt1/MDM2 pathway in breast cancer patients,these methods include homology modeling,structure and function prediction,Protein-Protein Interaction Analysis（PPI）,Molecular Dynamics（MD）simulation and trajectory analysis.5.BT-549 and He La cell lines were transfected with three kinds of recombinant plasmids: pc DNA^TM5-PIK3CA-WT,pc DNA^TM5-p.H1047 L and pc DNA^TM5-p.H1047 R.After a certain time of transfection,the cells were treated with 10μM TQ.WB was used to detect the downstream signal pathway after the cells were lysed with an appropriate amount of EBC buffer.6.After TQ treatment,the downstream signaling pathway of mutant group was significantly changed compared with WT group,so we conducted molecular docking study on TQ and PIK3 CA.Results: 1.PIK3 CA is highly conserved in different species,and its high expression is associated with low RFS in breast cancer,which proves that PIK3 CA plays a carcinogenic role in breast cancer patients.The frequency of PIK3 CA mutation was the highest（28.9%）among all the mutated genes in breast cancer patients.Among the PIK3 CA mutations,the frequency of H1047 mutation was the highest,with p.H1047 R and p.H1047 L at the top.2.The three recombinant plasmids were verified by PCR,double enzyme digestion and sequencing,and all of them could successfully express FLAG tag protein in cells,which proved that the cloning was successful.WB showed that the mutations could enhance the phosphorylation of Akt1 in BT-549 cells（the phosphorylation of p.H1047 R in BT-549 cells was slightly higher than that of p.H1047L）,but did not increase the total amount of Akt1.3.The band structure of PIK3CA/ΔNp63α complex suggests that the increase of p-Akt1 level may be due to the conformational change of PIK3CA/ΔNp63α complex caused by p.H1047 R and p.H1047 L mutations.PPI data showed that p.H1047 R can more effectively destroy the stability of PIK3CA/ΔNp63α complex than p.H1047 L.4.The number of amino acids and hydrogen bonds in PIK3CA/ΔNp63α complex will affect the interaction between PIK3 CA and ΔNp63α.MD simulation analysis showed that the conformational changes of p.H1047 R mutants were the most serious on the 20,30,40 and 50 ns time axes.Moreover,trajectory analysis also found that the fluctuation of p.H1047 R mutants was the most obvious after mutation.Principal Component Analysis（PCA）analysis,conformational entropy and Free Energy Landscapes（FEL）analysis further explained the increase of Akt1 phosphorylation in PI3 K pathway.5.TQ could effectively inhibit the expression or activate downstream proteins of PI3K/Akt1pathway: p-Akt1,MDM2,and phosphatidylinositol 3,4,5 triphosphate（PIP3）in the mutants（p.H1047 R,p.H1047L）.Molecular docking results of TQ and PIK3 CA showed that TQ could bind to PIK3 CA,and the binding site was around the 700-1000 amino acid residues of PIK3 CA.We speculated that TQ could exert its inhibitory effect by binding to the kinase domain of PIK3 CA.Conclusions: In terms of function,our results clearly confirmed that the two mutants p.H1047 R and p.H1047 L could reduce the inhibitory effect ofΔNp63α on PIK3 CA kinase domain,resulting in increased activity of PI3 K downstream signal,which observed by WB detection.Structurally,the interaction between the DNA binding domain of ΔNp63α（resid: 114-359）and the kinase domain of PIK3CA（resid: 797-1068）were partially destroyed after the mutations.The downstream pathway was obviously inhibited by TQ treatment in those two mutants of p.H1047 R and p.H1047 L.The docking of TQ with PIK3 CA indicates that TQ may play an important role by inhibiting the kinase domain of PIK3 CA.The successfully study of two hotspot mutations of PIK3 CA not only helps to explore the disease mechanism of metastatic breast cancer（MBC）patients,but also has great significance for researchers to develop new small molecule targeted therapy strategies.